The examination of the peripheral blood is an important aspect in evaluating the health of the individual. An important parameter within this examination is the differential white cell (or leukocyte) count. This test is presently performed in one of three ways. The most traditional method involves the preparation of a blood smear which is then stained via the Romanowsky method. This stain differentially colors the different constituents of whole blood. A technologist can enumerate the various leukocyte classes, typically neutrophils, lymphocytes, monocytes, eosinophils, and basophils by microscopic examination of the stained blood smear. As this manual differentiation is very labor intensive, substantial research and development effort has been expended to obtain an automated procedure for the examination of blood samples.
Recently multi-subpopulation leukocyte differential analysis has been routinely obtained with automated microscopes. These automated microscopes include the Hematrak series of products manufactured by Geometric Data (Wayne, Pa.) and the Diff 350 and 400 produced by Coulter Biomedical (Concord, Mass.). When these instruments are presented with a Romanowsky stained blood smear, using an image analyzing computer, these instruments can locate and classify white blood cells using visual classification criteria similar to those used by the human technician. These criteria typically include nuclear and cytoplasmic optical density, color, shape, and texture.
Another approach to automation of leukocyte differential involves the use of flow cytometry. In this procedure, blood cells in suspension are passed through a transducer and the cells are classified on the basis of some measurable parameter such as light absorption, light scatter or electrical impedance. The advantages of these flow cytometry systems compared to microscope-based systems include relatively high sample throughput and the ability to count larger number of cells per sample thereby reducing sampling noise. Commercial clinical flow cytometers have been limited, thus far, to three leukocyte subpopulation differential. Leukocytes are classified in subpopulations called granulocytes, monocytes and lymphocytes. The current three part differential instruments are based on either measurement of light scatter or electrical impedance. Light scatter instruments such as ELT 1500 of Ortho Instruments (Westwood, Mass.) classify white blood cell subpopulations on the basis of their light scattering characteristics measured at two different angles, typically termed "low" and "orthogonal." Electrical impedance instruments such as the S+ series of Coulter Electronics (Hialeah, Fla.) and Cell Dyn 2000 of Sequoia-Turner Corporation, (Mountain View, Calif.), classify leukocytes based on their volume following exposure to a volume modifying reagent.
Until recently, the only commercial clinical flow cytometry instruments capable of obtaining a five-subpopulation leukocyte differential were the H-6000 and H*1 of Technicon Instruments (Tarrytown, N.Y.) These instruments classify leukocytes by measuring light scatter and absorption after elaborate cytochemical staining of the blood cells. Since this staining process is relatively slow, the throughput of these instruments is compromised.
Cell-Dyn 3000 of Sequoia-Turner Corp. (Mountain View, Calif.) performs a conventional five subpopulation differential based only upon light scattering characteristics of unstained leukocyte cells. Since inherently slow cytochemical staining is not used, the sample throughput of this instrument is kept extremely high. The advances made possible by Cell-Dyn 3000 are largely the result of the combination of two innovations, namely measurement of depolarized orthogonal light scatter and the lytic agent of the present invention, enabling better resolution of lymphocytes and monocytes to obtain a five-subpopulation leukocyte differential analysis.
In commercially available automated instruments using flow cytometry, the leukocyte classification and differentiation is obtained either by light scatter or by electrical impedance related to cell size. In either type of instrument, the red blood cells in the whole blood sample must be lysed to release the hemoglobin. In electrical impedance measurement-based systems, quaternary ammonium-salt-based lysing agents have been used. These electrical impedance systems have an adequate response time which substantially nullifies the lytic effect of the lysing agent on the leukocytes. In light scattering measurement-based systems, the adverse effect of the red blood cell lysing agent upon the light scattering characteristics of the white blood cells is more severe, and thus more demanding from a design perspective. Lytic agents (used herein interchangeably with "lysing agents") must rapidly lyse red blood cells while simultaneously providing a window in which white blood cell light scattering characteristics are essentially undisturbed.
These criteria are typically met using a lytic agent with an alkaline pH, since the range of lysis of both red blood cells and white blood cells increases with pH. The widely used ammonium chloride/potassium bicarbonate/diNaEDTA lysis solution has a pH of 7.2-7.4, but typically takes 5-10 minutes to completely lyse red blood cells. Lytic agents such as Ortho Instruments, "Lyse Right" have a pH of .sup..about. 8.5 and require several seconds to effect complete lysis of red blood cells, but will adversely affect the light scattering characteristics of white blood cells within a period of a minute, or so. When the pH is raised much above this, or lowered below about 3, lytic agents are obtained that lyse both red blood cells and white blood cells almost instantaneously.
Therefore, it is an object of this invention to provide a lytic agent which adequately lyses red blood cells while preserving the light scattering characteristics of the white blood cells for the period of time adequate to obtain subpopulation differentiation.
It is a further object of this invention to provide a lytic agent which enables the enumeration of five leukocyte subpopulations, identified as neutrophils, lymphocytes, monocytes, eosinophils and basophils, in a flow cytometry, light-scattering system.
It is another object of this invention to provide a lyric agent which has a commercially acceptable shelf life.
These and further objects of the present invention will become apparent to those of skill in the art with reference to the specification and figures.